使用液相色谱-质谱联用技术对HMG-CoA还原酶抑制剂和表达调节剂进行基于细胞的筛选。

Cell-based screen of HMG-CoA reductase inhibitors and expression regulators using LC-MS.

作者信息

Gerber Raphaele, Ryan Jessica D, Clark Douglas S

机构信息

Department of Chemical Engineering, 201 Gilman Hall, University of California, Berkeley, CA 94720-1462, USA.

出版信息

Anal Biochem. 2004 Jun 1;329(1):28-34. doi: 10.1016/j.ab.2004.03.023.

DOI:10.1016/j.ab.2004.03.023

PMID:15136164

Abstract

We present an integrated consolidation of previously reported methods for screening hydroxymethylglutaryl-coenzyme A reductase (HMGR) inhibitors in 96-well microtiter plates with rapid workup using established mammalian cell lines and liquid chromatography-mass spectrometry analysis. Inhibitors as well as expression regulators of HMGR (inducers or repressors) can be screened. To validate the method, three competitive inhibitors of HMGR (lovastatin, simvastatin, and atorvastatin), as well as a potent sterol repressor of HMGR synthesis (25-hydroxycholesterol), were assayed on two cell lines: HepG2, a human hepatic derived cell line, and L cells, a subline of NCTC clone 929 mouse fibroblasts. The direct inhibition of HMGR by statins, induction of HMGR synthesis by the same statins following incubation with the cells, and repression of HMGR synthesis by 25-hydroxycholesterol were confirmed.

摘要

我们展示了一种整合方法，该方法结合了先前报道的用于在96孔微量滴定板中筛选羟甲基戊二酰辅酶A还原酶（HMGR）抑制剂的方法，并利用已建立的哺乳动物细胞系和液相色谱-质谱分析进行快速处理。HMGR的抑制剂以及表达调节剂（诱导剂或抑制剂）均可被筛选。为验证该方法，在两种细胞系上检测了三种HMGR竞争性抑制剂（洛伐他汀、辛伐他汀和阿托伐他汀）以及一种HMGR合成的有效固醇抑制剂（25-羟胆固醇）：HepG2，一种人肝源性细胞系；L细胞，NCTC克隆929小鼠成纤维细胞的一个亚系。证实了他汀类药物对HMGR的直接抑制作用、与细胞孵育后相同他汀类药物对HMGR合成的诱导作用以及25-羟胆固醇对HMGR合成的抑制作用。